1. Analytical Method Development and Validation
Bob Seevers

2. Importance of Analytical Methods in Development Stability Studies
Without analytical methods it is not possible to know what has happened during stability
Assay
Impurities/Degradation Products
Dissolution
Chiral Purity
Preservative Content

3. Interactive Role of Analytical Methods and Stability Data
This is a chicken and egg problem
Need analytical methods to generate stability data
Need stability information to develop methods
Start with preliminary method based on what is known of drug substance
Can determine content of drug substance
Known or expected degradants are separated
Use preliminary method to analyze stress stability studies
Stress deliberately creates significant degradation
Can evaluate ability of method to separate degradants
Use this information to modify/improve method

4. Types of Stress Studies
Photostability
High Temperature
Low Temperature
Oxidation
pH extremes

5. Selection of Chromatographic Mode
HPLC
Reversed-phase HPLC
Chiral HPLC GC
TLC
Electorphoresis

6. Identifying Degradants
Predicting routes of degradation
Acid/base hydrolysis of esters and amides
Oxidation of thiols, alcohols and amines
Loss of methyl groups
Synthesizing possible degradants
Prepare possible degradant
Known Structure
Test it in potential analytical method
Detectability
Separation from parent peak

7. Assay Run Time vs. Detection/Resolution
Long run times for an assay mean that stability testing will take more time and cost more
Shorter run times mean that testing will go faster and be less expensive but
Lower resolution
Some peaks may not be separated

8. Validation Protocol
A validation protocol lays out ahead of time the experimental design that will be used to establish the validity the analytical methods
Reagent, solvents
Sample, standard, solution preparation
Identify equipment to be used
Chromatographic conditions
System suitability
Calculations

9. ICH Q2 Validation of Analytical Procedures
Types of Analytical Procedures to be Validated
Identification tests;
Quantitative tests for impurities' content;
Limit tests for the control of impurities;
Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product.
See also USP General Chapter <1225>, Validation of Compendial Methods

10. With Regard to Stability
Types of Analytical Procedures to be Validated
Identification tests;
Quantitative tests for impurities' content;
Limit tests for the control of impurities;
Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product.
Drug product assay
This will also include quantifying dissolution results

11. Typical Validation Characteristics
Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness

12. Accuracy Closeness of agreement between the value found and either
the value accepted as a conventional true value
or an accepted reference value

13. Accuracy for Drug Substance
Several methods of determining accuracy are available:
application of an analytical procedure to an analyte of known purity (e.g. reference material);
comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure, the accuracy of which is stated and/or defined;
accuracy may be inferred once precision, linearity and specificity have been established.

14. Accuracy for Drug Product
Several methods for determining accuracy are available:
application of the analytical procedure to synthetic mixtures of the drug product components to which known quantities of the drug substance to be analysed have been added;
in cases where it is impossible to obtain samples of all drug product components , it may be acceptable either to add known quantities of the analyte to the drug product or to compare the results obtained from a second, well characterized procedure, the accuracy of which is stated and/or defined;
accuracy may be inferred once precision, linearity and specificity have been established.

15. Precision
Precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.
Repeatability
intermediate precision
reproducibility.
The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated.

16. Repeatability Repeatability should be assessed using:
a minimum of 9 determinations covering the specified range for the procedure (e.g., 3 concentrations/3 replicates each); or
a minimum of 6 determinations at 100% of the test concentration.

17. Intermediate Precision
Depends on the circumstances under which the procedure is intended to be used.
Look at effects of random events on the precision of the analytical procedure.
Days
Analysts
equipment, etc.
It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged.

18. Reproducibility
Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility should be considered in case of the standardization of an analytical procedure, for instance, for inclusion of procedures in pharmacopoeias. These data are not part of the marketing authorization dossier.

19. Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.
Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s).

20. Identity
Able to discriminate between compounds of closely related structures which are likely to be present.
The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the analyte.

21. Detection Limit
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
Can be determined
Visually
Signal to Noise
Standard Deviation of the Response and the Slope

22. Detection Limit: Signal to Noise
This approach can only be applied to analytical procedures which exhibit baseline noise.
Comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected.
A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

23. Detection Limit: Standard Deviation of the Response and the Slope
The detection limit (DL) may be expressed as: DL = 3.3 σ/S
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways, for example:
Based on the Standard Deviation of the Blank
Based on the Calibration Curve

24. Quantitation Limit
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.

25. Quantitation Limit Visual Evaluation Signal-to-Noise
Standard Deviation of the Response and the Slope
quantitation limit (QL) may be expressed as: QL = 10 σ/S
σ = the standard deviation of the response
S = the slope of the calibration curve
The estimate of σ may be carried out in a variety of ways, for example:
Based on the Standard Deviation of the Blank
Based on the Calibration Curve

26. Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.
Test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares.
correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted
minimum of 5 concentrations is recommended

27. Range
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
The following minimum specified ranges should be considered:
For the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration;
For content uniformity, covering a minimum of 70 to 130 percent of the test concentration, unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified;
For dissolution testing: +/-20 % over the specified range

28. Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
stability of analytical solutions;
extraction time.
influence of variations of pH in a mobile phase
influence of variations in mobile phase composition
different columns (different lots and/or suppliers)
Temperature
flow rate.

29. Method Validation Report
Provide results of validation effort
Explain
Choice of acceptance criteria
How method was developed
Method Validation Report will be submitted to regulators

30. Revalidation May be Necessary
Changes in the synthesis of the drug substance;
Changes in the composition of the finished product;
Changes in the analytical procedure.